The search for molecular targets/pathways that can be manipulated to improve bone properties is a highly active area of investigation. Recently, particular interest has been expressed in targeting biomolecules that can augment mechanical signaling in bone; the next generation of osteoporosis drugs is likely to work in conjunction with physical activity/loading in order to disproportionately direct new bone formation to skeletal areas that need it most (i.e., those regions that endure the greatest strains and are at the greatest risk of failure). The WNT signaling pathway has emerged as a key regulator of bone mass and strength, but also of bone cell mechanotransduction. Recent work by Professor Wim van Hul in Antwerp and by Novartis Pharma in Basel identified an accessory protein?LRP4?that regulates the activity of SOST, which is a secreted inhibitor of the WNT co-receptors LRP5/LRP6. Specific missense mutations in LRP4 cause high bone mass (HBM) phenotypes by eliminating SOST-mediated inhibition of LRP5/LRP6. The goal of the present application is to understand precisely how LRP4, LRP5, and LRP6 function to regulate bone metabolism and mechanotransduction, which ultimately will reveal new approaches for preventing or treating bone disorders?a primary mission of NIAMS/NIH. Among the key questions we addressed are: (1) Is the principal role of LRP4 in bone to bind SOST? (2) Does LRP4 directly present SOST to LRP5/LRP6 or simply increase local concentration of this inhibitor? (3) Does LRP4 function throughout osteoprogenitor differentiation, or does it have stage-specific roles? (4) Does LRP4 regulate LRP5 and LRP6 equally, or is LRP4 more important for one of these two WNT co-receptors? (5) Will bone-specific deletion/inhibition of LRP4 prevent the bone-wasting effects of mechanical disuse? (6) Does LRP4 coordinate the deposition of new bone to high-strain surfaces during mechanical loading? (7) Do LRP5 and LRP6 differ in terms of when (early vs. late in differentiation) and where (cortical vs. trabecular envelopes) they are active? (8) Which upstream and downstream mechanical signaling pathways will be identified in specific bone cell subtypes using single-cell transcriptomics of loaded and unloaded bone? We will use cutting-edge, novel, mouse models (CRISPR-based Lrp4 and Lrp6 knockins), single-cell transcriptomic approaches (Drop-seq and 10XGenomics), live cell microscopic techniques (FRET/FLIM), mechanotransduction models (strain mapping in ulnar loading and tail suspension), and radiographic/histologic/biochemical approaches to reveal the underlying biology and therapeutic potential of LRP4, LRP5, and LRP6 manipulation in bone tissue. The project is a continuation of the close collaboration between the Robling (Indiana Univ.) and Warman (Harvard Univ.) labs, an extremely fruitful partnership for more than 12 yrs. We have assembled a unique combination of expertise, resources, biological models/tools, and technical innovation to elucidate the role of LRP4 in bone biology.